To maintain tissue oxygenation, red cell formation from progenitors in bone marrow, spleen and fetal liver is strictly regulated (Baumann, R., and Dragon, S. 2005. Erythropoiesis and red cell function in vertebrate embryos. Eur J Clin Invest 35 Suppl 3:2-12). In early progenitors, erythroid lineage commitment is directed by a unique set of DNA binding and transcription factors (e.g., GATA-1, EKLF-1 and FOG-1) (Cantor, A. B., and Orkin, S. H. 2005. Coregulation of GATA factors by the Friend of GATA (FOG) family of multitype zinc finger proteins. Semin Cell Dev Biol 16:117-128). Subsequent pro-erythroblast expansion is likewise sharply controlled, in part, by the glycoprotein hormone erythropoietin (Epo) (Richmond, T. D., Chohan, M., and Barber, D. L. 2005. Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol 15:146-155). Epo is expressed in adult kidney via hypoxia-inducible transcription factor pathways (Richmond, T. D., et al., 2005. Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol 15:146-155). Epo's subsequent interactions with its single transmembrane receptor (EpoR) are then thought to selectively support erythroblast survival (Koury, M. J., and Bondurant, M. C. 1990. Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells. Science 248:378-381; Wu, H., Liu, X., et al., 1995. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 83:59-67; Socolovsky, M., et al., 1999. Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98:181-191). Redundant EpoR-activated survival pathways, in fact, have been described that depend upon phosphoinositide-3 kinase (PI3 kinase) and AKT-dependent regulation of Fox03a (Ghaffari, S., et al., 2006. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation. Blood 107:1888-1891) and (m)TOR (Levine, A. J., et al., 2006. Coordination and communication between the p53 and IGF-1-AKT-TOR signal transduction pathways. Genes Dev 20:267-275), as well as EpoR/Jak2/Stat5-dependent induction of Pim1 kinase (Hammerman, P. S., et al., 2005. Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. Blood 105:4477-4483) and the Bcl2 orthologue Bcl-xl (Socolovsky, M., et al., 1999. Cell 98:181-191). These response pathways therefore likely contribute in important ways to Epo's clinical utility as an anti-anemia agent and as an apparent cytoprotective factor for injured heart, endothelial, neuronal and renal cells (Maiese, K., Li, F., and Chong, Z. Z. 2005. New avenues of exploration for erythropoietin. Jama 293:90-95). Currently, research into Epo-mediated physiological processes such is hindered by the lack of a substantially homogeneous population of bone marrow derived Epo-responsive cells. Such a population of cells would be a boon to Epo-research.
Epo has also been recognized as a performance supplement by certain athletes. It is thought to help the athlete as an ergogenic aid by eliminating fatigue symptoms by increasing red blood cell count thereby increasing stamina and performance. However, Epo also has potential detrimental side effects for the athlete and has been banned by most if not all sports organizations. Unfortunately, testing for Epo doping by prior art methods is difficult, time consuming and/or inaccurate and complicated by the fact that the body makes physiological amounts of Epo (Scott J, Phillips G C., 2005, Erythropoietin in sports: a new look at an old problem. Curr Sports Med Rep. August; 4 (4):224-6).
Thus, what is needed are new reagents and methods useful for the investigation of signaling pathways and cellular processes involving Epo as well as for an efficient and accurate in vivo/ex vivo methods suitable for the detection of physiologically active Epo, Epo derivatives and Epo mimetics in individuals.